Nuclear Factor-kappaB (NF-kB) is an inducible transcription factor that plays an important role in a large number of cellular processes including immune and inflammatory responses, cell growth/differentiation, and cell survival. NF-kB activity is regulated primarily through its induced nuclear translocation, however additional signals are also required to enhance the ability of nuclear NF-kB to activate transcription. Many of these signals target the C-terminal transactivation domain (TAD) of the p65 subunit of NF-kB and likely function to enhance the association of transcriptional coactivator proteins with NF-kB. Several coactivator proteins are known to be important for NF-kB-dependent transcriptional activation including the CREB binding protein (CBP)/p300, p300/CBP associated factor (ptCAF), and members of the steroid receptor coactivator (SRC) family. In addition, it is also known that inducible phosphorylation of p65 is able to enhance the association of CBP with p65, supporting the idea that signals in addition to nuclear translocation play an important role in activation of NF-kB. Based on this, the main objective of the work described in this proposal is to gain a better understanding of the coactivator proteins that function with NF-kB to enhance its ability to activate transcription. This objective will be accomplished by performing the following three aims: AIM1 will address the role of protein arginine methyltransferase proteins in mediating NF-kB-dependent transcriptional activation; AIM 2 is focused on studying the role of the SRC-3 coactivator in mediating NF-kB-dependent transcriptional activation as well as correlating SRC-3 function with the IKK complex function as a coactivator; and in AIM 3 we have initiated a cytoplasmic two-hybrid screen in which we have identified several potential proteins that interact with the C-terminal TAD of p65. This AIM is focused on characterizing the functional interaction between these proteins and p65. Completion of these AIMs will provide critical insight into the basic mechanisms of NF-kB-mediated transcriptional activation.